HTMLE ▪ Chromate Fixatives

Chromic Acid Precipitates all proteins and adequately preserves carbohydrates

▪ Transition between dehydration and infiltration of embedding medium: Clearing
Regaud’s (Muller's) Fluid Chromatin, mitochondria, mitotic figures, golgi bodies, RBCs and colloid-
✓ FDD CIET SSML containing tissues
Orth's Fluid Study of early degenerative processes and tissue necrosis; Rickettsia and other
▪ Physical Methods of Fixation bacteria
✓ Heating (smears of microorganisms) Potassium Dichromate Preserves mitochondria, fixes lipids
✓ Microwaving (a form of heat fixation and is now widely practices in routine lab)
▪ Picric acid Fixatives
✓ Cryopreservation (Freeze drying)
Bouin's Solution Embryos and pituitary biopsies; GIT biopsies, animal embryos and endocrine
▪ Chemical Method of Fixation gland tissue
✓ Immersion fixation (immersing the specimen in the fixative solution) GREEN GREGORIOS: Preserving soft and delicate structures (endometrial
✓ Perfusion fixation (perfusing or injecting the fixative) curettings); Fragmentary biopsies; Masson’s trichrome; Not suitable for fixing
kidney structures, lipid and mucus
✓ Vapor form (used in specialized histochemical procedures)
Hollande’s Solution GIT specimens and endocrine tissues; Has decalcifying properties
Gendre’s Solution Glycogen and other carbohydrates
▪ Four Major Groups of Fixatives
✓ Aldehydes (formaldehyde, glutaraldehyde) – act by cross-linking proteins BLACK GREGORIOS: Preserving soft and delicate structures (endometrial
curettings); Fragmentary biopsies; Masson’s trichrome; Not suitable for fixing
✓ Oxidizing agents (osmium tetroxide, potassium permanganate) – act by cross-linking proteins
kidney structures, lipid and mucus
✓ Alcohol-based fixatives (methanol, ethanol, acetic acid) – act by denaturing proteins Brasil's Alcoholic Picroformol Best routine fixative for glycogen
✓ Metallic fixatives (mercuric chloride and picric acid) – act by forming insoluble metallic precipitates
▪ Other Fixatives
▪ Aldehyde Fixatives Glacial acetic acid (HAc) Nucleoproteins (nuclei); Chromosomes and chromatin materials (nuclear
10% Formol-Saline Best for CNS and Gen. post-mortem tissues, ideal for silver impregnation components); Destroys mitochondria and Golgi bodies
10% Neutral-Buffered Formalin Best Gen. tissue fixative; Frozen section, Iron and elastic fibers; Lead Fixatives Acid mucopolysaccharides and CT mucin
Immunohistochemistry and interphase FISH Trichloroacetic Acid (TCA) Proteins and nucleic acids; Weak decalcifying agent
Zinc Formalin (unbuffered) Immunohistochemistry Acetone Water diffusible enzymes (Lipases, Phosphatases); Brain tissues for rabies
Formol-Corrosive (F-Sublimate) Routine post-mortem tissues, Silver Reticulum methods; Neutral fats and diagnosis (Negri bodies); Solvent in freeze-substitution tech.
phospholipids Michel’s Solution Transport medium of fresh unfixed tissues; NOT a fixative
Paraformaldehyde EM; Immunocytochemical analysis; Paraffin embedding and sectioning
Karnovsky’s Fixative EM; Resin embedding and sectioning ▪ Recommend for Immunohistochemical techniques, cell smears, nucleic acids, CNS: Formaldehyde
Acrolein Electron Microscopy ▪ Recommended for chromaffin tissues, adrenal medulla and mitochondria: Chromate
Formol-calcium (Baker’s) Lipids (frozen section); Fixation & decalcification of calcium-rich tissues ▪ Recommended for Connective tissues and glycogen; Histones & basic proteins; Biogenic and polypeptide
Gendre’s Sputum cytology hormones Immunostaining; Fragmentary biopsies: Picric acid
Lillie’s (Calcium acetate formalin) Phospholipids ▪ Good fixative and excellent stain for lipids: Osmium tetroxide
Cajal’s Formol-ammonium bromide Nervous tissue (astrocytes)
▪ Very good for cytologic smears such as PAP smears (spray cans): Alcohol
Glutaraldehyde Made up of 2 formaldehyde residues, linked by a three- carbon chain
Histochemistry and Electron Microscopy ▪ Nuclear and histochemical fixative: Newcomer’s fluid
▪ Alcoholic Fixatives ▪ Microanatomical and histochemical: 10% Formol saline
Methyl Alcohol (100%) Wet and dry smears, blood smears, bone marrow tissues and cell cultures; Diluent ▪ Microanatomical and nuclear: Heidenhain’s susa
in Wright’s stain ▪ Microanatomical and cytoplasmic: Zenker-formol (Helly’s)
Isopropyl Alcohol (95%) Fixing touch preparations (impression smears)
Ethyl Alcohol (70-100%) Histochemistry especially enzyme studies; Sodium urate crystals in patients with ▪ 10% NBF Composition
gout; DNA fragments (PCR) ✓ 37-40% formaldehyde - 100 mL (10% = equivalent to 3.7-4% formaldehyde)
Carnoy’s Fixative (most rapid) Chromosomes, lymph glands, urgent biopsies; Brain tissues for rabies diagnosis ✓ DW - 900 mL
(Negri bodies); Nissl and cytoplasmic granules ✓ Sodium phosphate, monobasic - 4 g
Clarke’s Solution Frozen sections and smears; Nucleic acids
✓ Sodium phosphate, dibasic (anhydrous) - 6.5 g *anhydrous – no water / water-free
Alcoholic Formalin Fixation or post-fixation of large fatty specimens (breast); Lymph nodes detection
Formol-Acetic Alcohol Diagnostic cryostat sections ▪ Bouin’s Fixative Composition
Gendre's Fixative Sputum; Frozen sections; Immunoperoxidase studies and EM; Glycogen and ✓ 2.1% Picric acid saturated aqueous solution - 75 mL
Micro-incineration technique
✓ 40% formaldehyde - 25 mL
Newcomer's Fluid Mucopolysaccharides and nuclear proteins
✓ Glacial Acetic acid - 5 mL
▪ Mercuric chloride Fixatives
▪ Most commonly used fixative in histology: Formaldehyde
Mercuric chloride Tissue photography; Renal tissues, fibrin, CT and muscles; Metachromatic staining
and Trichrome staining; Hematopoietic & RES ▪ Most widely used fixative for routine histology: 10% NBF
Zenker's Solution Good general fixative; Recommended for PTAH & Trichrome staining; Congested ✓ Fixation temperature: RT (18-30°C)
specimens (lung, heart and blood vessels) ✓ Normally used: 20-22°C
Zenker-Formol (Helly’s) Solution Blood-containing organs (pituitary gland, BM, liver and spleen); Extramedullary
hematopoiesis and intercalated discs of cardiac muscle ▪ Fixative Osmolality
Lillie’s B-5 Fixative BM biopsies (hematopoietic tissues); Immunohistochemical staining ✓ Best results are obtained using slightly hypertonic solutions (400-450 mOsm)
Heidenhain's Susa Solution Tumor biopsies (skin) ✓ Hypertonic solutions = Shrinkage
Schaudinn's (Sublimated alcohol) Wet smears for cytologic examinations
✓ Isotonic (340 mOsm)/Hypotonic solutions = swelling and poor fixation
Ohlmacher's Solution Lipids and proteins; Nervous tissue and brain; Nuclear preservation
Carnoy-Lebrun Solution Lipids and fatty tissues (liver and adipose); Nuclear preservation ✓ Added to Osmium tetroxide fixatives for EM: Sucrose
▪ Fixative pH: Neutral (6.0-8.0)
▪ Osmium tetroxide Fixatives
Flemming's Solution Nuclear structures (Chromosomes); Fixes fats ▪ The Optimum temperature range for fixation is: 37-56°C
Flemming's w/o Acetic Acid Cytoplasmic structures (mitochondria) ✓ Moderate Heat: Accelerates fixation but hastens autolytic changes and enzyme destruction
▪ Traditional/usual: RT (18-30°C) Normally used: 20-22 ▪ Rapid acting, recommended for urgent biopsies and routine purposes; Carcinogenic or may damage bone
▪ Tissue processors: 40-42°C marrow (Aplastic anemia): Benzene
▪ Electron Microscopy and Histochemistry: 0-4°C ▪ Recommended for CNS tissues and cytological studies (esp. Smooth muscles and skin): Cedarwood oil
▪ Mast cells for EM: RT ▪ Recommended for clearing embryos, insects and very delicate specimens: Aniline oil
▪ Used to slow down decomposition if the tissue needs to be photographed and cannot be fixed ▪ Its quality is not guaranteed due to its tendency to be adulterated; Not suitable for routine purposes
immediately: Refrigeration because it is expensive: Clove oil
▪ Nucleic acids fixation: Rapid at higher temp.
▪ Rapid fixation of very urgent biopsy specimens: Formalin heated to 60°C ▪ Clears both Paraffin and Celloidin sections; Quality is not always uniform and good and is extremely slow:
▪ Fixing tissues with tuberculosis: Formalin heated to 100°C Cedarwood oil
▪ Dehydrates and clears at the same time since it is miscible in both water and paraffin: Tetrahydrofuran
▪ Used to prevent formation of dark brown artifact pigment granules (hematin) when using formalin fixatives:
▪ Properties are very similar to chloroform but it is cheaper; Toxic on prolonged exposure:
✓ Add buffer
Carbon tetrachloride
Sat. alcoholic Picric acid
▪ Slow-acting clearing agents that can be used when double embedding techniques are required:
Others: Acidified potassium phosphate buffer, Sodium acetate buffer and Citrate buffer
Methyl-benzoate/Methyl-salicylate
✓ Add Alcoholic KOH (1% KOH in 80% alcohol)
▪ Fixative and decalcifying agent: Trichloroacetic acid, Picric acid, Chromic acid ▪ The clearing agent that becomes milky on prolonged storage and its quality is not always good and uniform:
▪ Fixative and dehydrating agent: Alcohol (Methanol, Carnoy’s, Gendre’s), Acetone Cedarwood oil
▪ Fixative and stain: Picric acid, Osmium tetroxide ▪ The clearing agent that becomes milky when dehydration is not complete: Xylene
▪ Decalcifying agent and tissue softener: Perenyi’s fluid
▪ Infiltrating and embedding media:
▪ Dehydrating agent and clearing agent (universal rgt): Tetrahydrofuran, Dioxane, 3° Butanol, Oil of Bergamot
✓ Paraffin
▪ May cause excessive hardening or brittleness of tissues: ✓ Paraffin substitutes
✓ Prolonged fixation ✓ Celloidin
✓ Prolonged dehydration ✓ Gelatin
✓ Prolonged clearing ✓ Resin
✓ Prolonged infiltration
✓ Prolonged embedding (overheating) ▪ The simplest, most common, and by far the best embedding medium for routine use: Paraffin wax
▪ Airholes found in the tissue during trimming is due to: Incomplete infiltration ▪ The impregnation method used for delicate specimens and frozen tissue sections because it prevents
▪ Prolonged dehydration in the higher grades of alcohol will render the specimen: Hard and brittle fragmentation of tough and friable tissue: Gelatin Impregnation
▪ Makes the tissue opaque and difficult to cut due the presence of alcohol: Incomplete clearing ▪ Specimens with large cavities or hollow spaces: Celloidin wax impregnation
▪ EM; Hard tissues (undecalcified bone); renal and BM biopsies: Resin/Plastic Impregnation
▪ Most common dehydrating agent /dehydrant: Ethanol
▪ Most common and fastest decalcifying agent: Nitric acid ▪ Celloidin Methods
▪ Decalcifies and softens tissues at the same time: Perenyi’s fluid a. WET Celloidin ▪ Recommended for bones, teeth, large brain sections and whole organs.
▪ Dehydration is accomplished through the use of: Increasing grades of alcohol Method ▪ Tissues must be cut wet (both the knife & tissue block are kept moist with 70%
✓ Starting Concentration: 70% Alc. while cutting).
✓ Final Concentration: 100% b. DRY Celloidin ▪ Preferred for processing of whole eye sections.
Method ▪ Material embedded with the dry method can be cut without alcohol due to
▪ The most common (routine) and most rapid decalcifying agent used so far: Nitric acid the presence of the cedarwood oil in the block.
▪ Best for cellular preservation and staining; Recommended for routine decalcification of post-mortem ▪ Gilson’s mixture – equal parts of chloroform and cedarwood oil.
research tissues: Formic acid
▪ Recommended for Surface decalcification of tissue blocks if used in 1% solution with 70% alcohol: ▪ Infiltration in overheated paraffin above ____°C produces shrinkage and hardening of tissues; Paraffin oven
Hydrochloric acid must be maintained at a temperature ____°C above the melting point of the wax. 60°C; 2-5°C
✓ Von Ebners (36% Sat. Aq. NaCl, Conc. HCl, DW) ✓ Ideal MP of infiltrating agent: 56-58°C
▪ Permits good nuclear staining; Does not require washing out; Not recommended for urgent examinations ✓ Normally used for routine work: 56°C (56-60°C)
(very slow acting): Trichloroacetic acid
▪ The most ideal, accurate, sensitive and reliable method to determine the end point of decalcification: ▪ The steps involved in automatic tissue processing using Autotechnicon
Radiological / X-ray ✓ It makes use of 12 individual processing steps, 10 for the reagents (1L) and 2 paraffin.
1. Fixation Container 1: Fixative
▪ Ratio of decalcifying agent to tissue volume: 20:1 Container 2: Fixative
✓ FDDCI = 20,20,10,10,25 2. Dehydration Container 3: Dehydrant
▪ The most commonly used and is considered as an excellent and true clearing agent: Xylene Container 4: Dehydrant
▪ A good clearing agent must be ____________ with dehydrating agent (alcohol), melted paraffin wax and Container 5: Dehydrant
mounting medium. Miscible Container 6: Dehydrant
3. Clearing Container 7: Clearing agent
▪ An excellent and true clearing agent; most rapid: Xylene Container 8: Clearing agent
▪ Substitute for xylene or benzene: Toluene Container 9: Clearing agent
▪ It is recommended for tough (skin, fibroid and decalcified tissues) and large tissue specimens; Best for Container 10: Clearing agent
nervous tissue, lymph nodes, granulation tissue, and fetal and other delicate, highly cellular specimens: 4. Infiltration Container 11: Melted or liquid paraffin
Chloroform Container 12: Melted or liquid paraffin
▪ Types of Microtomes ▪ The knife is usually tilted at ____° angulation on a microtome to allow a clearance angle between the cutting
SLIDING ▪ For cutting celloidin embedded sections facet and the tissue block. 0-15
▪ Base-Sledge Microtome and Standard Sliding Microtome
▪ MOST DANGEROUS because of its movable exposed knife (moved backward and forward) ▪ The color of the inside surface of floating-out water bath: Black
ROTARY ▪ For cutting paraffin embedded sections ✓ Purpose: For easier visualization of creases and folds in sections
▪ MOST POPULAR and MOST COMMON type used for routine and research studies
▪ Microtome inside the CRYOSTAT ▪ The thermostat of Floating-out bath should be set at ____°C, i.e. about ____°C below the melting point of the
ROTARY ▪ For cutting paraffin embedded sections wax used for blocking out. 45(45-50); 10 (6-10)
▪ MOST POPULAR and MOST COMMON type used for routine and research studies
▪ Microtome inside the CRYOSTAT ▪ Immediate separation of tissue from wax in a flotation water bath is typically caused by:
ROCKING / ▪ THE SIMPLEST ✓ Insufficient or incomplete paraffinization
CAMBRIDGE ▪ For cutting serial sections of large blocks of paraffin embedded tissues ✓ Inadequate wax quality or melting point
FREEZING ▪ For cutting unembedded frozen sections
✓ Incorrect water bath temperature
ULTRATHIN ▪ For cutting specimens into extremely thin slices for electron microscopy work
✓ Tissue not properly trimmed or oriented
▪ Inventors ▪ If a tissue section detaches from a slide:
MICROTOME ▪ Wilhelm His, Sr. (1865/66) - Swiss anatomist and professor ✓ Return it to the floatation bath to rehydrate and reattach
SLIDING ▪ Alexander Cummings (1770s) - invented the first practical Sliding Microtome
✓ Gently agitate the bath to help reposition the section
▪ George Adams (1789)
ROTARY ▪ George Richards Minot (1885-1886) ▪ Purpose of heat and xylene prior to staining after sectioning: Deparaffinization and drying of slides
ROCKING / ▪ Paldwell Trefall (1881)
CAMBRIDGE ▪ The type of staining technique employed in routine H and E staining: Regressive staining
FREEZING ▪ John Queckett (1848) ▪ Modified H & E: Progressive staining
CRYOSTAT ▪ Sir James Dewar (1897) REGRESSIVE The tissues are overstained and the excess dye is then removed until the desired intensity
ULTRATHIN ▪ Germans (1800s) is obtained
▪ Cold ultramicrotomy: Humberto Hernandez Moran PROGRESSIVE Staining is continued in a definite sequence until the desired intensity of coloring of the
different tissue elements is attained
▪ 3 MAJOR PARTS OF MICROTOME:
No washing / differentiation / decolorization
✓ Block holder - where the tissue is held in position DIRECT/SIMPLE The staining of tissue by means of simple alcoholic/aqueous solution of the dye
✓ Knife and knife carrier - for actual cutting of tissue sections (Methylene blue and Eosin)
✓ Pawl, Ratchet Feed Wheel and Adjustment Screws - to line up the tissue block in proper position with the SPECIFIC It is the basis of Histochemistry
It is accomplished by controlled, specific chemical reactions designed to give a final color
knife, adjusting the proper thickness of the tissue for successive sections
(staining) at the site/location of the structure of the substances in the cells or tissues
▪ Venetian blinds effect in histopathology refers to: COUNTERSTAINING The application of a different color or stain to provide contrast and background to the
staining of the structural components to be demonstrated.
✓ Characteristic striped or blinds-like pattern
✓ Caused by a damaged or nicked microtome blade INDIRECT The action of the dye is intensified by some other agents such as mordant and accentuator
METACHROMATIC Entails the use of the specific dyes that stain tissues with a color that is different from that of
▪ Thickness of sections METACHROMASIA the stain color itself
✓ Paraffin section: 4-6µ VITAL The selective staining of living cell constituents, demonstrating cytoplasmic structures by
✓ Celloidin section: 10-15µ phagocytosis of the dye particle (Cytoplasmic Phagocytosis)
✓ Freezing: 4µ INTRAVITAL Done by injecting the dye into any part of the animal body (either intravenous,
intraperitoneal or subcutaneous) producing specific coloration of certain cells, particularly
✓ Ultrathin: 60-100 nm; Semithin: 0.5-1 um those of RES (Lithium, India ink, Carmine)
▪ Cutting Stroke SUPRAVITAL Used to stain living cells immediately after removal from the living body (neutral red)
✓ Generally, a slow, uniform cutting stroke produces the best results and the least compression. ▪ A substance that serves as a link or bridge between the dye and the tissue to make the staining reaction
✓ Hard tissues (cervix or thyroid) are best cut with a firm, relatively quick stroke possible: Mordant
✓ Soft tissues are best cut with slow, gentle motion ✓ Without it ripened hematoxylin is almost useless because of its inherent low affinity for the tissue itself
▪ HONING ✓ Ehrlich’s hematoxylin: Potassium alum with Hematoxylin
✓ Removal of gross nicks and irregularities from the knife ✓ Weigert’s hematoxylin: Iron
✓ To remove blemishes and grinding the edge of the knife to acquire an even edge
▪ A chemical substance that does not participate but merely increases or heightens the color intensity,
✓ Heel-to-toe (edge first)
crispness and selectivity of the stain: Accentuator
✓ Hones (sharpening stones)
✓ Loeffler’s Methylene Blue: Potassium hydroxide
Carborundum Hones = for badly nicked knives
✓ Carbol thionine and carbol fuchsin: Phenol
Arkansas Stone = medium fineness
Yellow Belgian/Belgium Yellow = finest (best result) ▪ The complex of dye/stain and mordant is called a: Lake
Belgian Black Vein = oil stones
▪ Agents usually used for ripening of stains:
▪ STROPPING ✓ Sodium iodate
✓ Removal of “burr” formed during honing and polishing the cutting edge of the knife ✓ Mercuric Oxide
✓ Toe-to-heel (edge last) ✓ Potassium permanganate
✓ Paddle strop made up of horse leather ✓ Calcium hypochlorite
✓ Hydrogen peroxide
▪ The surface of the hone is wiped clean with a soft cloth moistened with xylene in order to remove the
scattered small particles of stones and metal. It is then covered with a thin film of Mineral and Clove Oil, Xylene, ▪ Commonly used for the rinsing and bluing steps of the H&E staining: Tap water
Liquid Paraffin or Soapy Water for: Lubrication ✓ Used for alkalinization; Alkalinity is a measure of water's ability to neutralize acid
 ✓ EXAMPLE: The alkaline pH of the bluing solution causes the mordant-dye (lake) to reform in the tissue and ▪ Helicobacter pylori (H. pylori), the following stains are commonly used:
become more permanent. ✓ Giemsa stain = Widely used and effective
✓ Warthin-Starry stain (silver nitrate) = gold standard
▪ Bluing agents ✓ Steiner stain
✓ 1% Ammonia water (Ammonium hydroxide water) ✓ Genta stain (modified Giemsa)
✓ 1% Lithium carbonate (dilute) ✓ Immunohistochemistry (IHC) for H. pylori antigens
✓ Scott’s tap water (water, potassium carbonate, magnesium sulfate)
✓ Scott’s TWS (Tap water substitute) ▪ Romanowsky stains are a group of polychromatic stains used in cytology and histology. Examples include:
✓ 0.2-0.5% Bicarbonate ✓ Giemsa stain
✓ Potassium or Sodium acetate ✓ Wright's stain
✓ Potassium or Sodium hydroxide ✓ Leishman's stain
✓ May-Grünwald-Giemsa (MGG) stain
▪ The end point of a bluing agent ✓ Jenner-Giemsa stain
✓ Reached when the desired blue color is achieved, indicating complete neutralization of excess stain or ✓ Diff-Quik stain
acidity.
▪ The Feulgen stain is used to stain:
▪ Counterstains ✓ DNA
NUCLEAR CYTOPLASMIC ✓ Nucleic acids
RED RED ✓ Nuclear material (chromatin, nucleoli)
▪ Carmine ▪ Eosin Y = most common
▪ Neutral red ▪ Eosin B ▪ Hematoxylin stains:
▪ Safranin ▪ Phloxine B ✓ Nuclei (DNA)
BLUE ▪ Rose Bengal Resulting Stain: Purple-blue (dark blue, blue-black)
▪ Methylene blue YELLOW ✓ Nucleoli
▪ Toluidine blue ▪ Picric acid ✓ Chromatin
▪ Celestine blue ▪ Orange G
▪ Hematoxylin = most common GREEN ▪ Romanowsky stains (Giemsa, Wright's, MGG) are commonly used to assess the adequacy of fluid aspirates,
✓ Excellent: Ehrlich’s Hematoxylin ▪ Lissamine Green including:
▪ Light Green SF ✓ Blood smears
✓ Bone marrow aspirates
▪ The oldest of all stains and is used for staining amyloid, cellulose, starch, carotenes and glycogen: Iodine
✓ Cytological preparations (e.g., fine-needle aspirates)
▪ A dark green to black powder belonging to the phenazine group of dyes used to stain mitochondria in living
tissues: Janus green B ▪ Perl's Prussian Blue Reaction for Ferric iron
▪ Stains acid mucopolysaccharides; More specific for connective tissue and epithelial mucin: Alcian blue ✓ Solution: Acid ferrocyanide
▪ An excellent stain for Elastic fibers: Orcein 1% aqueous potassium ferrocyanide (20 mL)
▪ For observing cell granules and vacuoles of phagocytic cell: Neutral red 2% aqueous hydrochloric acid (20 mL)
▪ Used for demonstration of neuroglia in frozen sections: Victoria blue
▪ Turnbull's Blue Reaction for Ferrous Iron
▪ PAS positive substances ✓ Solution: Acid ferricyanide Solution:
✓ Carbohydrates (Glycogen, Glycoproteins, Mucins, Polysaccharides, Glycolipids) 1% aqueous potassium ferricyanide (20 mL)
✓ Glycoproteins (Immunoglobulins, Complement components, CRP, Amyloid) 2% aqueous hydrochloric acid (20 mL)
✓ Mucoproteins (Acidic mucins, Neutral mucins, Hyaluronic acid, Chondroitin sulfate)
▪ Stains commonly used to visualize Spirochetes:
✓ Phospholipids, Glycolipids, Unsaturated Lipids
✓ Warthin-Starry Stain: Black / dark brown (golden yellow background)
✓ Fungal cell walls (e.g., Aspergillus, Candida)
✓ Levaditi Stain: Black / dark brown (yellowish brown background)
✓ Bacterial capsules (e.g., Klebsiella, Pseudomonas)
✓ Giemsa Stain: Pinkish-red to purple / purple-blue (pale blue or purple background)
▪ PAS positivity in various tissues ▪ Lysochromes (Oil Soluble Dyes)
✓ Liver (glycogen) ✓ SBB - most sensitive and specific reagent for lipid staining; Greatest affinity for phospholipids & neutral fats
✓ Pancreas (glycogen) ✓ Sudan IV (Scharlach R) - Recommended for triglycerides (neutral lipids)
✓ Salivary glands (mucins) ✓ Sudan III - First Sudan dye introduced into Histochemistry; A good fat stain for CNS tissues
✓ Respiratory tract (mucins) ✓ ORO – stains neutral lipids and fatty acids in smears, cryostat sections and tissues
✓ Intestinal tract (mucins)
▪ Not lysochromes, but used to stain lipids
▪ Stains used to identify atherosclerotic plaques: ✓ Osmic acid, Nile blue sulfate
✓ Sudan dyes (lipids, fatty acids, and cholesterol)
✓ Oil Red O Stain (lipid accumulation - red) ▪ The most commonly used impregnation agent for demonstrating nervous tissues and reticulin: Silver nitrate
✓ H and E stain (visualize the general morphology) ▪ A vegetable dye derived from lichens, commonly used for staining elastic fibers: Orcein
✓ Masson's Trichrome Stain (collagen fibers and fibrous cap)
✓ Von Kossa Stain (calcium deposits – dark brown to black) ▪ An old histologic dye extracted from the female Cochineal Bug (Coccus cacti): Cochineal
✓ Alizarin Red S (calcium deposits – red) ✓ Carmine - used as a powerful chromatin and nuclear stain for fresh material and smear preparations
✓ PAS stain (carbohydrates and mucopolysaccharides) ✓ Picrocarmine - used in neuropathological studies
✓ Alcian Blue Stain (acidic mucopolysaccharides) ✓ Best Carmine - used for demonstration of glycogen
▪ For calcium salts and phosphatase activity: Acridine red 3B ▪ Components of Pap Stain
▪ A basic acridine fluorochrome which permits discrimination between dead and living cells, giving GREEN ✓ Hematoxylin= nuclear stain (basic)
fluorescence for DNA and a RED fluorescence for RNA: Acridine Orange ✓ Counterstains:
▪ For demonstrating calcium involves replacement of the anionic part of calcium salts with silver. Silver is made (1) Orange Green-6 (OG-6) = cytoplasmic stain (acidic)
visible following reduction by exposure to bright light: Von Kossa - Staining mature cells (Mature superficial cells).
▪ Used in frozen sections for rapid diagnosis: Loeffler’s PMB - Stains keratin (bright intense orange) and was originally used to stain the small cells of keratinizing
squamous cell carcinoma present in sputum.
▪ Mounting Media (Mountants)
- Phosphotungstic acid, a mordant, strongly binds to protein and helps to intensify the color achieved.
AQUEOUS RESINOUS
▪ Water ▪ Canada Balsam (2) Eosin Azure-50 (EA-50) = cytoplasmic stain (acidic)
▪ Glycerin ▪ D.P.X. - Staining immature cells (Intermediate and parabasal cells)
▪ Farrant’s medium/Gum syrup ▪ Xam Eosin Y stains - Stains the superficial epithelial squamous cells, nucleoli, cilia, and RBCs
▪ Apathy’s medium ▪ Clarite Light Green SF yellowish - Stains the cytoplasm of other cells, including non-keratinized squamous
▪ Brun’s fluid ▪ Eukit cells; Quite expensive and difficult to obtain
▪ Entellan Alternative: Fast Green FCF (produces visually different results)
▪ Aqueous Mountants are usually made up of: Bismarck brown - Stains nothing and in contemporary formulations it is often omitted.
✓ Gelatin, glycerin jelly or gum arabic - to solidify the medium ▪ Not recommended for cytologic smears because it is intensely stained by the basic Light Green counterstain
✓ Glycerol - to prevent cracking and drying of the preparation of Papanicolaou method: Egg albumin
✓ Sugar - to increase the refractive-index
▪ When specimen is more than a few drops, the fluid should be centrifuged first. This is usually done at
✓ Preservative solution
moderate speed at _____ RPM for ___ minute(s). 2000 RPM, 2 minutes
▪ Recommended for mounting frozen sections from water or paraffin sections that require dehydration and ▪ By far the best method to collect cells from body fluid (e.g., urine, pleural or peritoneal fluid):
clearing: Brun’s fluid ✓ Cytospin Preparation
▪ Does not solidify upon storage, eliminating the need for heating before use: Farrant’s / Gum syrup (RI: 1.43) ✓ 1000 RPM for 1 minute
▪ High refractive index; Also used as preservative: Glycerin (RI: 1.47)
▪ General purpose aqueous mountant; Used for Methylene Blue-stained nerve preparations: Apathy’s (RI:1.52) ▪ Two major methods used to obtain high-quality respiratory tract specimens from patients with suspected
▪ Recommended for mounting frozen sections from water or paraffin sections which require dehydration and pulmonary tuberculosis (TB) but a sputum-scarce or smear-negative status: BAL and BW (Bronchial washing)
clearing: Brun’s (RI:1.43) ▪ Usually performed in patients with AIDS to rule out P. jirovecii (carinii): BAL (Bronchoalveolar Lavage)
▪ Low refractive index; good only for temporary mounting: Water (RI:1.333)
▪ In vaginal hormonal cytology, the smears are taken from the: Upper lateral third of the vaginal wall
▪ Process of sealing the margins of the coverslip to prevent escape of fluid or semi-fluid mounts & evaporation ▪ Cervical mucus exhibits “palm-leaf” pattern: Ferning phenomenon
of mountant, to immobilize the coverslip, and to prevent sticking of slides upon storage: Ringing ✓ This occurs because of Salt crystal formation
✓ Kronig Cement - Made up of 2 parts Paraffin wax mixed with 4-9 parts powdered Colophonium resin ✓ It indicates high persistence of Estrogen
✓ Used as a basis for Early pregnancy
▪ Fixative for EM
✓ Primary fixatives: Osmium tetroxide, glutaraldehyde, paraformaldehyde the procedure is performed at 4°C ▪ Advantages of Liquid-based cytology (LBC)
✓ Karnovsky’s paraformaldehyde-glutaraldehyde, Acrolein (glutaraldehyde/formaldehyde); Platinic chloride ✓ Cleaner background
and Zamboni’s ✓ Less time required to read the slide
✓ Increased sensitivity and specificity
▪ Electron Microscopy (EM) Stains ✓ Improved sample quality and reduced unsatisfactory samples
✓ Uranyl acetate: Provides higher contrast for general EM imaging
✓ Lead citrate: Enhances contrast for biological samples ▪ Tiny pieces of unrelated tissue that can interfere with biopsy interpretation: Floaters
✓ Ruthenium red: Stains polysaccharides, glycoproteins, and acidic mucins ✓ Originate from:
✓ Toluidine blue: Stains cartilage, bone, calcified tissues, mast cells, glycoproteins, and mucins Other patients' samples
Laboratory personnel
▪ The best fixative for cytological specimens requiring fixation: Ether alcohol (Ether and Ethanol - 1:1 ratio) Environmental sources
Previous biopsy samples
▪ The best fixative for exfoliative cytology: Ether alcohol ✓ Lead to:
Misdiagnosis
▪ Most commonly used: 95% Ethanol
Incorrect staging
▪ Fixative for all types of effusion: 50% Alcohol Unnecessary treatments
Delayed or missed diagnosis
▪ Used as alternative fixatives for cytological smears
▪ Grossly normal lungs when placed in a solution will: Float
✓ Hair spray (contains PVP, alcohol and polymers)
✓ Resolution: Cover the lungs with several layers of gauze
✓ Corn syrup & Honey (high sugar content, viscosity and antimicrobial properties)
✓ Spray cans of alcohol ▪ Filing for the MTLE typically opens how many months before the exam date? 3 months
✓ Methanol and 95% ethanol ✓ Duration: 2 months Deadline: 1 month before the exam
Exam date: March 21-22, 2024
▪ The best method for routine cytologic examination: Papanicolaou method Opening of online processing: December 21, 2023
▪ Second best: Phase-contrast microscopy Deadline for applications: February 21, 2024
▪ The Expanded Newborn Screening (ENBS) Program (RA 9288) includes: PRE-TREATMENT OF TISSUE SECTIONS
✓ Congenital Hypothyroidism (CH) Antigenic determinants masked by formalin-fixation and paraffin embedding often may be exposed by
✓ Phenylketonuria (PKU) epitope unmasking, enzymatic digestion or saponin, etc. Do not use this pretreatment with frozen sections or
✓ Galactosemia (GAL) cultured cells that are not paraffin-embedded.
✓ Congenital Adrenal Hyperplasia (CAH) PROCEDURE
✓ Glucose-6-Phosphate Dehydrogenase Deficiency (G6PD)
1. Rinse sections in PBS-Tween 2 times for 2 minutes each time
✓ Congenital Thalassemia PBS = Phosphate buffered saline, pH 7.4
▪ Attending a seminar is an example of: 2. Serum Blocking: Incubate sections with normal serum block-species same as secondary antibody, for 30
✓ Formal learning: Structured and planned (seminars, conferences, courses and institutional training) minutes to block non-specific binding of immunoglobulin
✓ Non-formal learning: Structured and unplanned (self-study, online tutorials like YT, workshops w/o cert)
Purpose: To block unwanted non-specific staining. Sources include endogenous enzymes, fluorochromes,
✓ Informal learning: Unstructured and unintentional (life or observational experiences, interactions & convos) endogenous antibody binding activity and cross reactivity of the secondary reagents with endogenous proteins
▪ The best way to communicate patient results to a provider is: Electronic communication Common blocking buffers: Normal serum, not-fat dry milk, BSA or gelatin, and commercial blocking buffers
Note: This protocol uses avidin-biotin detection system. Avidin-biotin block may be needed based on tissue type
✓ Ensures accuracy, legibility, timeliness, documentation and security (HIPAA compliance)
3. Primary Antibody: Incubate sections with primary antibody at appropriate dilution in primary antibody
▪ A Z-score represents: dilution buffer for 1 hour at room temperature or overnight at 4°C
✓ The number of standard deviations a data point is from the mean of a dataset
Target antigens: Proteins that are within or on the surface of a cell
✓ A Z-score of 0 indicates the value is exactly at the mean
Primary antibody: Binds specifically to the target antigen
✓ A positive Z-score indicates the value is above the mean Direct IHC: Primary antibody is directly conjugated to the label
✓ A negative Z-score indicates the value is below the mean
4. Rinse in PBS-Tween 20
▪ Wound from friction or rubbing against a rough surface: Abrasion (open wound)
5. Peroxidase Blocking: Incubate sections in peroxidase blocking solution for 10 minutes at room
▪ Cut or tear in the skin from a sharp object: Laceration (open wound)
temperature
▪ Caused by a sharp, pointed object that penetrates the skin: Puncture wound (open wound)
▪ Blood collection outside blood vessels due to trauma: Hematoma (closed wound) Purpose: To prevent potential distraction of endogenous peroxidase activity; to eliminate false positive reactions
Peroxidase blocking: Absolute methanol containing 0.5% hydrogen peroxide for 10 minutes at RT
▪ Bruise from blood leakage into tissues after trauma: Contusion (closed wound)
ALP blocking: 1 mm concentration of levamisole
▪ Animals used for production of monoclonal antibodies: Mouse
6. Rinse in PBS-Tween 20
▪ Polyclonal antibodies: Rabbit, goat, pig, sheep, horse, guinea pig
7. Secondary Antibody: Incubate sections with biotinylated secondary antibody at appropriate dilution in
▪ The first step in immunohistochemistry: Fixation PBS for 30 minutes at room temperature
▪ A preparation step prior to staining that only applies to immunohistochemistry: Antigen retrieval
Secondary antibody: Binds to the primary antibody
▪ Antigens are most likely to be demonstrated at max sensitivity with: Frozen sections fixed in acetone Indirect IHC: Secondary antibody is conjugated to the label
✓ Preparing tissue for immunohistochemistry: In certain instances, the tissue must be prepared as a cryostat 8. Rinse in PBS-Tween 20 3 times for 2 minutes each time
section and fixed for a few seconds in absolute methanol or acetone, to preserve immunological activity
and prevent destruction of some of the labile antigenic sites. However, immunofluorescence and immuno- 9. Detection: Incubate sections in streptavidin-HRP in PBS for 30 minutes at room temperature
peroxidase techniques may also be done on formaldehyde-fixed and paraffin embedded sections. Enzyme labels: Horseradish peroxidase (HRP), Alkaline phosphatase (ALP)
▪ Immunohistochemistry (IHC) 10. Rinse in TBS (Tris-buffered saline) 3 times for 2 minutes each time
✓ Labels: Enzymes (HRP, ALP)
11. Chromogen/Substrate: Incubate sections in DAB solution for 1-3 minutes
✓ Visualization: Bright field microscopy
Purpose: It forms an insoluble colored precipitate that can be visualized under a microscope
▪ Immunofluorescence (IF)
Common Chromogens:
✓ Labels: Fluorescent dyes (FITC, TRITC) • 3,3’ diaminobenzidine (DAB) = dark brown end products
✓ Visualization: Fluorescence or confocal microscopy • 3-amino-9-ethylcarbazole (AEC) = red end products
▪ In IHC, many masked antigens can now be retrieved in routinely processed tissue by: Note: Should be made fresh immediately before use; Add hydrogen peroxide, failure to add results in total lack of
✓ Proteolytic enzyme digestion staining of all cell slides, including positive controls
✓ Microwave antigen retrieval Advantage of DAB chromogenic staining:
✓ Microwave The colored precipitate is NOT sensitive to light and the slides can be stored for many years
Optimal incubation time for linking antibodies with peroxidase conjugates: 30-60 minutes (RT)
✓ Trypsin antigen retrieval
✓ Pressure cooker antigen retrieval 12. Rinse in PBS-Tween 20 2 times for 2 minutes each time

▪ Horseradish peroxidase (HRP) and alkaline phosphatase (ALP), which convert 3,3' diaminobenzidine (DAB) 13. Counterstain if desire
and 3-amino-9-ethylcarbazole (AEC), into _____ and _____ end products, respectively. Brown and red Purpose: Provides a contrast to the chromogen and also helps the pathologist visualize the underlying tissue
structure
▪ A relatively new technique that involves the boiling of formalin-fixed deparaffinized sections in certain Most common: Hematoxylin (Blue background; Stains nuclei blue)
solutions, such as 0.01 M Citrate/Citric acid buffer (pH 6.0), EDTA (pH 8.0) or Tris EDTA (pH 10.0): Microwave
14. Rinse in distilled water
antigen retrieval
15. Dehydrate through 95% ethanol for 2 minutes, then 100% ethanol for 2 times 3 minutes each time
▪ Used in rapid fixation, rapid embedding, special staining and antigen retrieval in IHC: Microwave processing 16. Clear in xylene
17. Coverslip with mounting medium
▪ Microwave works as a physical agent to increase the movement of molecules and accelerate fixation.
✓ Also used to accelerate staining, decalcification, IHC and EM Formaldehyde is good for immunohistochemical techniques. The standard solution is 10% neutral buffered
✓ Major advantage: tissue is heated right through the block in a very short time formalin or approximately 3.7%-4.0% formaldehyde in phosphate buffered saline.
▪ Malignant Neoplasms: referred to as Cancers, derived from “cancrum”, a Latin word for crabs Pathologist: from a list submitted by PSP
✓ Sarcoma – Mesenchymal / Connective tissues 2 RMTs: from a list submitted by PAMET
✓ Carcinoma – Epithelial tissues ✓ Term of office = 3 years
✓ Can only be removed by the President of the Philippines
▪ Carcinoma
✓ Malignant tumor of Epithelial tissue (skin or tissues around internal organs) ▪ Administrative investigation: Revocation or suspension of COR
✓ Done by at least 2 members of the board + 1 legal officer
▪ Sarcoma
Revocation = 3 out of 3 votes (unanimous)
✓ Malignant tumor of Connective tissue (Bone, cartilage, fat, muscle, blood vessels or other connective or
Suspension (not more than 2 years) = 2 out of 3 votes (majority)
supportive tissue)
✓ COR must be surrendered within 30 days after the decision is made (the suspension period shall begin
▪ Adenoma from the date of such surrender.
✓ Benign tumor of Epithelial tissue
▪ The Professional Regulation Commission (PRC) or Komisyon sa Regulasyong Pampropesyonal
✓ Derived from the ducts and acini of glands, although the name is also used to cover simple tumors
✓ A three-man commission attached to Department of Labor and Employment (DOLE).
arising in solid epithelial organs
✓ Composed of 1 Chairperson and 2 Commissioners
▪ Papilloma ✓ Appointed by: the President of the Philippines
✓ Benign tumor of Epithelial tissue ✓ Term of office: 7 YEARS (R.A. 8981)
✓ Take origin from an epithelial surface ✓ Chairperson: Charito A. Zamora
▪ Leiomyoma - Benign tumor of the Striated muscle ✓ Commissioners: Jose Y. Cueto, Jr and Erwin M. Enad
▪ Rhabdomyoma - Benign tumor of the Smooth muscle (heart muscles) ▪ PAMET
▪ Myosarcoma - Malignant tumor of the Muscle ✓ National organization of all MTs
✓ Rhabdomyosarcoma - Malignant tumor of the Striated muscle (skeletal muscles) ✓ The only Accredited Professional Organization (APO) of Registered Medical Technologists
✓ Leiomyosarcoma - Malignant tumor of the Smooth muscle ✓ Organized by Crisanto G. Almario, Father of PAMET (9/15/63 at the Public Health Laboratory in Sta. Cruz
Manila)
▪ Potentially explosive reagents
✓ 9/20/64 = 1st national convention (FEU)
✓ Picric acid, silver salts, sodium azide, dioxane
✓ Charlemagne Tamondong = 1st president
▪ Values included in the Code of Ethics: Reliability, honesty and integrity ✓ Nardito Moraleta = 2nd president
▪ Core Values of PAMET: Integrity, Commitment, Excellence, Unity, Professionalism ✓ Current president: Luella A. Vertucio
▪ PAMET Hymn Lyrics: Hector Gentapanan Gayares, Jr. ▪ PASMETH: The Philippine Association of Schools of Medical Technology and Public Health, Inc.
▪ PAMET Hymn Music: Francis Jerota Pefanco ✓ The national organization of all registered schools of medical technology in the Philippines; Formed in
1970
▪ Memo No. 2012-0154
✓ 6/22/70 = 1st organizational meeting (UST)
✓ Inclusion of Maple Syrup Urine Disease (MSUD) in the New Born Screening Testing, since MSUD is the
✓ 5/7/71 = 1st annual meeting (UST)
most common inborn error of metabolism in the country.
✓ Dr. Gustavo Reyes = 1st president
▪ T test ✓ Current president: Dr. Jose Jurel M. Nuevo
✓ Measures accuracy; Comparison of means of 2 groups of data
▪ Nardito Moraleta
▪ F test ✓ Professionalized the MT course (Professional Recognition)
✓ Measures precision; Comparison of SDs of 2 groups of data ✓ Worked for the approval of RA 5527
✓ Wrote the 1st MT code of ethics on 8/6/68z
▪ Classifications of Medical Laboratories (based on service capability)
▪ Emergence of the Profession; 1st PAMET President
✓ Primary Laboratory - A clinical laboratory based on service capability which includes:
✓ Charlemagne Tamondong
Routine Hematology (CBC) - Hgb conc, Hct vol. conc
▪ Professional Recognition
WBC Differential Count
✓ Nardito Moraleta
Qualitative platelet determination
Routine Analysis and fecalysis RA 5527 Philippine Medical Technology Act of 1969 (6/21/69)
Gram Staining & Blood Typing RA 6138 Amended RA 5527 (8/31/70)
✓ Secondary Laboratory - Includes the primary lab services and the following: (1st Amendment) Sections 16, 21, 22
Routine Clinical Chemistry- blood glucose, BUN (blood urea nitrogen), BUA (Blood Uric Acid), PD 498 Amended RA 5527 (6/28/74)
Creatinine & total cholesterol (2nd Amendment) Sections: 2, 3, 4, 7, 8, 11, 13, 16, 17, 21, 29
Qualitative platelet determination PD 1534 Amended RA 5527 (6/11/78)
Cross Matching (3rd Amendment) Sections 3, 8, 11 and 13
Gram staining Amended PD 498 – section 12
KOH (for fungal disease) RA 8504 An act promulgating policies and prescribing measures for the prevention
✓ Tertiary Laboratory - Includes the secondary lab services and the following and control of HIV/AIDS in the Philippines
Special Chemistry- Cardiac markers RA 9288 Newborn Screening Act of 2004
Special Hematology- Coagulation test RA 5527 An Act Requiring the Registration of Medical Technologists
Immunology/Serology- HIV, hepa profile, tumor markers RA 9165 An act instituting the comprehensive dangerous drugs act of 2002
Microbiology (C/S) RA 6425 The dangerous drugs act of 1972
▪ Board of Medical Technology (1 pathologist, 2 RMTs) RA 8981 An act modernizing the Professional Regulation Commission
✓ Chairman: Dr. Marilyn A. Cabal-Barza RA 6969 An act to control toxic substances and hazardous and nuclear wastes
✓ Members: Dr. Leila Lany M. Florento and Ma. Lourdes L. Gatbonton RA 4688 Clinical Laboratory Law
✓ Appointed by the President of the Philippines RA 1517 Blood Bank Law of 1956
 RA 7719 National Blood Services Act of 1994 ▪ Excellent substitute for hone or stone: Plate Glass
RA 11166 Philippine HIV and AIDS Policy Act of 2018 ▪ Best screening marker for Lymphoma: Leukocyte Common Antigen (LCA) / CD45
RA 11223 Universal Health Care Act
EO No. 266 Institutionalization of the Continuing Professional Education (CPE) programs AUTOMATION
CMO No. 6 series of 2008 Guidelines for the Accreditation of Clinical Laboratories involved in the
training of Medical Laboratory Science/Medical Technology Interns ▪ Tissue Processing:
✓ Sakura (Tissue-Tek VIP, Tissue-Tek Prisma)
AO No. 2007-0027 Revised Rules and Regulation governing the Licensure and Regulation of
✓ Leica (ASP300, ASP600)
Clinical Laboratories ✓ Thermo Scientific (STP 420)
AO No. 2008-0007 Schedule of Fees for the Licensure of General Clinical Laboratories and the ✓ Milestone (Tissue Processor)
Registration of Special Clinical Laboratories
AO No. 2008-0008 Rules and Regulations Governing the Regulation of Blood Services Facilities ▪ Digital Pathology:
CMO No. 14 series of 2006 Policies, Standards and Guidelines for Medical technology Education ✓ Olympus (VS120, VS200)
✓ Hamamatsu (NanoZoomer, S360)
Luella A. Vertucio Current PAMET President ✓ Philips (IntelliSite, Ultra-Fast Scanner)
Gamaliel A. Fulgueras Current PAMET Vice President ✓ Leica (Aperio, AT2)
Dr. Jose Jurel M. Nuevo Current PASMETH President
Dr. Ferdinand A. Mortel Current PASMETH Vice President ▪ Vacuum Infiltration Processor (VIP)
✓ Sakura Finetek (Tissue-Tek VIP series)
Dr. Marilyn A. Cabal-Barza Current MedTech Board Chairman
✓ Leica Biosystems (ASP series)
Dr. Leila Lany M. Florento Current Board Members ✓ Thermo Scientific (STP series)
Ma. Lourdes L. Gatbonton
Charito A. Zamora Current PRC Chairperson ▪ Automated Embedding Systems
Teofilo Pilando Jr. Past PRC Chairperson ✓ Sakura Finetek (Tissue-Tek Embedding Center; autoTEC)
Jose Y. Cueto, Jr Current PRC Commissioners ✓ Leica Biosystems (EG1150, EG1160)
Erwin M. Enad ✓ Thermo Scientific (Shandon Embedding Center)
✓ Milestone (Tissue Embedding System)
▪ RA 5527 Penal Provisions:
✓ Any person who shall practice Medical Technology without registration or exemption ▪ Automated Staining Systems
✓ Any MT who shall knowingly make a fraudulent laboratory report ✓ Sakura Finetek (Tissue-Tek DRS 2000)
✓ Roche Ventana (BenchMark ULTRA, HE 600)
✓ Any MT who practices without supervision of pathologist/physician
✓ Leica Biosystems (ST4040, ST4080, Bond-III)
✓ Any MT who failed/refused to display COR at workplace ✓ Dako (Omni, Autostainer 360)
▪ COR may be revoked or suspended due to penal provisions and also for the following reasons: ✓ Biocare (Medical Care, CareDx)
✓ Agilent (Dako Autostainer Plus)
✓ Unprofessional conduct
✓ Malpractice and Incompetency ▪ Automated Coverslipping Systems:
✓ Serious ignorance or negligence in the practice ✓ Sakura Finetek (Tissue-Tek Film Coverslipper)
✓ Violation of ethical standards ✓ Leica (CV5030, CV5032)
✓ Conviction of a crime involving moral turpitude ✓ Thermo Scientific (Shandon CoverSlipper)
✓ Non-compliance with continuing education requirements ✓ Dako (Coverslipper)
✓ BioGenex (i6000 Coverslipper)
▪ Nerves cannot be donated due to complex neural connections, risk of neurological damage and limited
regeneration capacity ▪ Automated Staining and Coverslipping Systems
✓ Sakura Finetek (Tissue-Tek Prisma)
▪ Serosa is the outermost layer of an organ, a thin membrane surrounding abdominal organs (e.g., intestine, ✓ Leica Biosystems (ST4040, ST4080)
stomach), thoracic organs (e.g., heart, lungs) and reproductive organs (e.g., ovaries, testes) ✓ Dako (Omni, Autostainer 360)
✓ Roche Ventana (HE 600, BenchMark ULTRA)
▪ The cortex is the outer layer of an organ with specific functional and structural roles, as found in the adrenal ✓ Agilent (Dako Autostainer Plus)
gland, kidney, and brain
▪ Laboratory Information Systems (LIS):
▪ Routine fixative in Histopathology: 10% NBF
✓ Cerner (CoPathPlus)
▪ Best fixative for EM and IHC: Karnovsky's Paraformaldehyde-Glutaraldehyde ✓ Epic (Beaker)
▪ Excellent fixative for Glycogen preservation: Alcohol ✓ McKesson (Pathology)
▪ Best decalcifying agent for cellular preservation and staining: Formic Acid ✓ Sunquest (CoPath)
▪ Best dehydrating agent; most common: Ethanol
▪ Best dehydrating agent for EM: Ethanol ▪ Immunohistochemistry (IHC) Automation:
✓ Roche Ventana (BenchMark ULTRA)
▪ Used as a transition fluid: Propylene dioxide
✓ Leica (Bond-III, Bond-MAX)
▪ Substitute for Ethanol: Isopropanol ✓ Dako (Omni, Autostainer 360)
▪ The best clearing agent for Microwave Technology: Isopropanol ✓ Biocare (Medical Care)
▪ Best embedding medium: Paraffin wax
▪ Best Vital dye/Supravital stain: Neutral Red ▪ Other Automation Solutions:
▪ Best stain for EM: Uranyl Acetate ✓ Agilent (Automated DNA extraction)
▪ Excellent Connective Tissue (CT) fibers stain: Mallory's Trichrome, Masson’s Trichrome ✓ Qiagen (Automated nucleic acid extraction)
▪ Excellent Nuclear stain: Ehrlich's Hematoxylin ✓ BioGenex (Automated IHC, ISH)
▪ Best honing tool: Belgian Yellow (or Belgium stone)